We are studying the organization of mitochondria during the development of the hamster preimplantation embryo. The development of preimplantation embryos from many species including the hamster is suboptimal in vitro. Recent improvement in the ability to culture embryos has occurred largely because of efforts to understand energy substrate requirements. Mitochondrial activity which is an integral part of preimplantation embryo metabolism, may be adversely affected by inappropriate culture conditions. Multiphoton microscopy with the fluorescent probes Rhl23, NAO and Mitotracker will be used to investigate (1) the distribution of active mitochondria versus all mitochondria at specific points throughout embryogenesis in the hamster and (2) the effects of culture conditions on the localization and activity of mitochondria in the hamster embryos. The use of multiphoton microscopy will be crucial for this study as we require very low levels of phototoxicity in order to maintain the viability of the the embryos. The information gathered will help in the understanding of the regulation of metabolism of the preimplantation hamster embryo and ultimately result in improved ability to support embryo development in vitro.